Project Summary/Abstract: Breast cancer is the most common cancer in women, with an estimated 246,660 cases (and 40,450 deaths) in the US during 2015. Due to better screening techniques cancers are caught earlier and 75% of patients are candidates for breast conserving surgery (BCS) to remove the cancer. BCS is cosmetically preferable to the alternative (mastectomy) and long-term survival rates are equivalent [1]. The success of BCS is assessed post-operatively by pathology. The status of the microscopic margins of excision of the lumpectomy specimen is still the most important prognostic and risk factor for local recurrence [2,3]. A positive margin indicates that invasive carcinoma or ductal carcinoma in situ is touching a tissue edge of a lumpectomy specimen. Among patients treated by BCS and radiation therapy, positive margins are associated with a 2-fold increase in the risk of local recurrence when compared with negative margins [4]. A finding of positive margins is estimated to occur between 8% to 86% of the time, requiring patients to return for further treatment often associated with poorer cosmetic results and increases in local and distant recurrence of the disease. Current pathology methods only assess about 1/10 of 1% of the entire volume of the removed specimen. A consequence of margin undersampling is that local recurrence occurs in 5-16% of patients with pathologically clean margins, suggesting that one or more regions of tumor had not been sampled during pathological analysis resulting in tumor remaining in the patient. In addition, there is still no universal agreement among breast surgeons on what constitutes an adequate negative margin for patients undergoing BCS [5]. Together these data demonstrate the unmet clinical need for technologies that rapidly and globally identify cancerous tissues in the surgical cavity and can be used to guide their surgical resections during the procedure. Molecular imaging guided resections of tumors during surgeries are now being developed. However, most approaches employ IV administration of optical imaging agents, which require hours or days to highlight tumor tissues. Moreover, infiltrating cancer cells in tissues surrounding the main mass may not have developed a vasculature and likely would not be identified using injected agents. Finally, illuminating the entire cancer mass may create a high background signal from tumor that is not ?at the margin? of the lumpectomy. Exploiting increased protease expression at the edge of breast cancers we introduce the novel concept of in vivo topical administration of quenched fluorescent molecular imaging probes to identify cancer that may remain in the surgical cavity after standard-of-care resection. This builds on years of preclinical studies and seeks to characterize, perform toxicology, and finally a Phase 1A & B clinical trial to demonstrate the utility of this novel approach. It has the potential to reduce re-excisions as well as the false negative rate from pathology undersampling, with a consequent savings in healthcare costs and, enhancement in patient life quality.